Ganglioside biosynthesis in Golgi apparatus: new perspectives on its mechanism.

The synthesis of gangliosides GM1 and GM2 in intact rat liver Golgi vesicles is stimulated by phosphatidylglycerol as much as or even more than by detergents (Triton X-100 and octyglucoside, respectively). The antibiotic tunicamycin, known as an inhibitor of the N-glycosylation of proteins, strongly inhibits the synthesis of the above gangliosides, in the presence as well as in the absence of the phospholipid. Both phosphatidylglycerol dependence and tunicamycin inhibition disappear when the Golgi vesicles are solubilized by addition of detergents or disrupted by ultrasonication or pretreated with pronase. Transport studies with UDP-[3H]Gal show that tunicamycin blocks the penetration of the sugar nucleotide into the Golgi vesicles in a concentration-dependent manner up to 80%. The results show that tunicamycin inhibits ganglioside biosynthesis by blocking the transport of the nucleotide sugar and not by inhibiting the transferase directly. Studies on glycoprotein-galactosyltransferase with ovalbumin as exogenous acceptor showed that phosphatidylglycerol does not destroy the integrity of the Golgi vesicles. So this phospholipid is an excellent tool for studying ganglioside biosynthesis at optimal transferase activities without solubilizing the Golgi membranes.